Reverse transcription is the process by which a retrovirus such as HIV-1 converts its single-stranded (ss) RNA genome into a double-stranded DNA copy that is integrated into host chromosomal DNA. This process is complex and is catalyzed by the virion-associated enzyme, reverse transcriptase (RT). However, another viral protein, the nucleocapsid protein (NC), is also required for productive viral DNA synthesis. (A) We study the mechanistic basis for NC activity. HIV-1 NC is a small (7 kDa), basic nucleic acid binding protein with two zinc fingers (ZFs), each containing the invariant CCHC Zn-coordinating motifs. It is also a nucleic acid chaperone, i.e., NC facilitates remodeling of nucleic acid structures so that the most thermodynamically stable conformations are formed. This property is critical for promoting efficient and specific reverse transcription. (i) Recent studies have focused on Gag C-terminal cleavage products i.e., NCp15 (NCp9-p6), NCp9 (NCp7-SP2), and mature NC (NCp7), and comparison of their nucleic acid chaperone activities in reconstituted systems modeling early reverse transcription events, i.e., annealing and elongation steps in tRNALys3-primed (-) strong-stop (SS) DNA synthesis and subsequent minus-strand transfer. The maximum levels of annealing are similar for all of the proteins, but there are important differences in their ability to facilitate RT-catalyzed DNA extension. Thus, at low concentrations, NCp9 has the greatest activity, but with increasing concentrations, DNA synthesis is significantly reduced. This finding reflects NCp9s strong aggregating activity and nucleic acid binding affinity (associated with the highly basic C-terminal SP2 domain), which together with its slow dissociation kinetics, limit the ability of RT to traverse the nucleic acid template during DNA elongation. NCp15 has much lower minus-strand transfer activity than NCp9 or NCp7, which we attributed to NCp15's acidic C-terminal p6 domain. Indeed, mutants with Ala substitutions in the acidic residues have improved chaperone activity. Viewed collectively, our findings help to further explain why complete processing of the NC precursors is critical for long-term virus replication and fitness. (ii) During synthesis of (-) strong-stop (SS) DNA, the RNase H activity of RT cleaves viral RNA, generating small 5-prime terminal RNA fragments that remain annealed to the DNA. These fragments must be removed so that minus-strand transfer can proceed. To test whether fragment removal is facilitated by NC destabilization of the short duplex and/or by RNase H cleavage, we used an NC-dependent system that models minus-strand transfer. The presence of short terminal fragments pre-annealed to (-) SSDNA had no impact on strand transfer, implying efficient fragment removal. Moreover, in reactions with an RNase H-minus RT mutant, NC alone was able to facilitate fragment removal, albeit less efficiently than in the presence of both RNase H and NC. Direct measurement of RNA fragment release from a duplex in the absence of DNA synthesis demonstrated that the architectural integrity of NC's ZF domains is absolutely required for this reaction. These findings are in excellent agreement with our earlier studies of the tRNA removal step in plus-strand transfer (Wu et al. 1999) and the ability of NC to block mispriming during initiation of plus-strand DNA synthesis (Post et al. 2009). Thus, HIV-1 uses a common mechanism for the RNA removal reactions required for successful reverse transcription. (iii) Comparative studies of the nucleic acid chaperone activities of simian immunodeficiency virus (SIV) and HIV-1 NCs have been initiated. Our findings thus far indicate that HIV-1 NC activity is about two-fold greater than that of SIV NC in reconstituted systems modeling the reactions involved in minus-strand transfer. Moreover, both HIV-1 and SIV NCs show very similar quantitative interactions with DNA in single molecule DNA stretching experiments, reflecting similarity of the domain structure of the two proteins. Further experiments are in progress. (B) Our interest in host proteins that affect HIV-1 reverse transcription and replication has led us to investigate the activities of human APOBEC3 (A3) proteins. (i) We have been studying A3A, which like other A3 family members, is a cytidine deaminase that converts dC residues to dU in ssDNA and functions as a DNA mutator. A3A inhibits a wide range of viruses, including retroviruses, and also displays potent activity against endogenous retroelements such as LINE-1. Our collaborators have determined the solution structure of A3A at high resolution using NMR spectroscopy. We have now performed structure-guided mutagenesis studies designed to characterize A3As enzymatic, nucleic acid binding, and biological activities. We show that A3A binds and deaminates ssDNA in a length-dependent manner. Surprisingly, although A3A also binds ssRNA, NMR analysis demonstrates that the RNA and DNA binding interfaces differ. Moreover, no deamination of ssRNA is detected in real-time NMR assays. In experiments on LINE-1 retrotransposition, assays with active- and non-active site A3A mutants reveal that the absence of deaminase activity per se does not always result in loss of anti-LINE-1 activity, demonstrating that these two activities are not linked. We have also performed experiments that indicate a mechanism for A3A's recently reported ability to mutate normally double-stranded genomic DNA, an activity that is implicated in carcinogenesis. Taken together, our studies provide new insights into the molecular properties of A3A and its role in multiple cellular and antiviral functions. (ii) In earlier work (Iwatani et al. 2007), we found that A3G inhibits RT-catalyzed HIV-1 DNA elongation reactions in a deaminase-independent manner due to its strong nucleic acid binding affinity and slow dissociation kinetics. We proposed that the inability of RT to traverse the RNA or DNA template creates a roadblock (hence the name roadblock mechanism). Recent single-molecule DNA stretching experiments provide support for this mechanism and show that A3G initially binds ssDNA with rapid on-off rates (compatible with enzymatic activity) and then subsequently converts to a slowly dissociating nucleic acid binding protein as A3G slowly oligomerizes on the viral genome, thereby inhibiting reverse transcription. In contrast, oligomerization-deficient A3G does not exhibit the slow off rate, but is still catalytically active. (iii) Current work is also focused on identifying the determinants of human A3H cytidine deaminase and anti-HIV-1 activities, using site-directed sequence- and structure-guided mutagenesis. We constructed a homology model of A3H based on the known structure of the C-terminal domain of A3G. The model reveals a large cluster of basic residues that are likely to be involved in nucleic acid binding and not surpringly, mutation of these residues to acidic or neutral amino acids results in reduction or in most cases, abrogation of enzymatic activity. Mutations in a structural element that dictates substrate specificity also negatively impact catalytic function. Interestingly, as shown for A3G, some of the A3H mutants that are defective in catalysis (including the catalytic mutant E56A) are able to restrict HIV-1 replication, although at a lower efficiency than wild type. This result raises the possibility that A3H inhibits HIV-1 by deaminase-dependent and -independent mechanisms. Endogenous reverse transcription assays with E56A mutant virions showing reduced synthesis of viral DNA and the fact that A3H can multimerize strongly suggest that a roadblock mechanism might also be relevant to A3H deaminase-independent HIV-1 restriction.